Cardiac cell culture material

ABSTRACT

The purpose of the present invention is to provide a cardiac cell culture material which specifically acts on cardiac cells. In addition, another purpose of the present invention is to provide artificial organ material obtained by culturing by using said cardiac cell culture material, and a method for producing the same. Thus, provided is a cardiac cell culture, wherein functional cardiac tissue is favorably built by using a cardiac cell culture material containing VCAM-1.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of International ApplicationPCT/JP2015/050028, filed on Jan. 5, 2015, and designated the U.S., andclaims priority from Japanese Patent Application 2014-142804 which wasfiled on Jul. 11, 2014, the entire contents of each of which areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a cardiac cell culture material and acell culture substrate on which a wall surface and/or a bottom surfaceof the culture substrate having the wall surface and/or the bottomsurface are coated with the cardiac cell culture material. In addition,the present invention relates to an artificial organ material obtainedby culturing a cardiac cell by using the cardiac cell culture material,and a method for producing the same.

BACKGROUND ART

Fibroblasts exist in almost all of vertebrate, and when tissue isinjured by trauma and ischemia, the injured area is replaced withfibrous tissue in accordance with fibroblasts proliferation and theabundant extracellular matrix deposition. Likewise, in a variety ofheart disease such as myocardial infarction and cardiomyopathies, a lotof cardiomyocytes were lost and also fibrous tissue replaces that area,which leads to cardiac remodeling and heart failure accompanied withexcess hemodynamics stress and neurohumoral stimulation. Althoughneurohumoral factors such as angiotensin II and endothelin-1 are wellknown to contribute to promote the cardiac remodeling via blood pressureelevation, cardiomyocyte apoptosis and local inflammation, cardiacfibroblasts have been reported to secrete those factors. Cardiacfibroblasts are also known to play a critical role in heartdevelopments. Interconnected cellular processes in a cardiac fibroblastform a network of collagen, fibroblasts and myocytes. Althoughcardiomyocyte proliferation is indispensable process of formation ofthick ventricular wall and embryonic cardiac fibroblasts have also beenreported to promote myocardial mitotic activity through β-1 integrinsignaling. The cardiac fibroblasts dominant causative substance has beenunclear. Herein cardiac fibroblasts multifariously act on heartdevelopment and pathogenesis and the importance of understanding ofmutual interaction and underlying mechanisms between cardiomyocytes andcardiac fibroblasts have been widely recognized. However the uncertainproperties of cardiac fibroblasts were the bottle-neck for it and it isrequired to reveal functional and molecular biological characteristicsof cardiac fibroblasts.

Heart tissue engineering is promising methods for not only regenerativemedicine, but also tissue models. Among cardiac tissue engineeringmethods, cell sheet-based cardiac tissue using temperature responsiveculture dishes have been developed. Previously, it was reported thatlayering of cardiac cell sheets containing neonatal rats-derivedcardiomyocytes, fibroblasts and endothelial cells on the various typesof vascular bed enabled to fabricate three-dimensional vascularizedviable cardiac tissue (Non patent documents 1 to 3). Since cellsheet-based tissue engineering does not need any scaffold, it requiressome amounts of extracellular matrices to construct cell sheets.Consistent with the evidences that left ventricle is mainly composed offibroblasts and cardiomyocytes, some amounts of fibroblast areindispensable to fabricate cardiac cell sheets when using purifiedembryonic stem cell-derived cardiomyocytes (Non patent document 4).Since recent reports have suggested that cell-cell interaction betweencardiomyocytes and non-myocytes is important for heart physiology andpathogenesis (Non patent document 5), fibroblasts function might alsoaffect the function of the engineered cardiac tissue and it might beprerequisite to select the suitable fibroblasts to fabricate the cardiactissue in vitro for tissue models. However it remains unclear whethercardiac fibroblasts have the specific function for cardiomyocytescompared with other types of fibroblasts and the related molecularmechanisms.

As mentioned above, since the cardiac fibroblasts play an important rolein heart developments, and the onset or cure of heart diseases, it isrequired to separate cardiac fibroblasts that specifically act oncardiac cells such as cardiomyocytes from other fibroblasts, and tosample the cardiac fibroblasts. According to the recent studies, it hasbeen revealed that fibroblasts, which were previously considered as auniform cell type, have a great variety of phenotypes, and that thephenotypes differ depending on a load state of existing organs, tissuesor cells.

However, the function of fibroblasts is not clearly known, andfibroblasts are only cells morphologically classified. Therefore, amongfibroblasts, it is difficult to select only one type thereof having aspecific function.

Meanwhile, with respect to vascular cell adhesion molecule-1 (VCAM-1,CD106) and α4 integrin, Kwee, et al. reported that VCAM-1 was expressedon embryonic day 11.5 at epicardium, cardiomyocytes, ventricular septum,and the like. It was also reported that, although the expression of α4integrin was recognized at similar areas as those of VCAM-1, α4 integrinwas not expressed in ventricular septum (Non patent document 6).Moreover, it was reported that, on embryonic day 11.5, there areembryonic death resulting from inhibition of formation of the placenta,and deformity due to decrease in dense layers of ventricular myocardiumand ventricular septum in an embryo that is defective in VCAM-1. Yang,et al. also reported an epicardium defect in α4 integrin null embryo ofembryonic day 11.5 (Non patent document 7). Accordingly, it isconsidered that VCAM-1 and α4 integrin mainly contribute to formation ofcardiac cells and epicardium in the embryonic stage.

CITATION LIST Non-Patent Document

-   Non-Patent Document 1: Shimizu T, et al., Fabrication of pulsatile    cardiac tissue grafts using a novel 3-dimensional cell sheet    manipulation technique and temperature-responsive cell culture    surfaces. Circulation research. 2002; 90:e40-   Non-Patent Document 2: Sekiya S, et al., Bioengineered cardiac cell    sheet grafts have intrinsic angiogenic potential. Biochemical and    biophysical research communications. 2006; 341:573-582-   Non-Patent Document 3: Shimizu T, et al., Cell sheet engineering for    myocardial tissue reconstruction. Biomaterials. 2003; 24:2309-2316-   Non-Patent Document 4: Matsuura K, et al., Hagiwara N, Zandstra P W,    Okano T. Creation of mouse embryonic stem cell-derived cardiac cell    sheets. Biomaterials. 2011; 32:7355-7362-   Non-Patent Document 5: Deschamps A M, et al., Disruptions and    detours in the myocardial matrix highway and heart failure. Current    heart failure reports. 2005; 2:10-17-   Non-Patent Document 6: Kwee L, et al., Defective development of the    embryonic and extraembryonic circulatory systems in vascular cell    adhesion molecule (vcam-1) deficient mice. Development (Cambridge,    England). 1995; 121:489-503-   Non-Patent Document 7: Yang J T, et al., Cell adhesion events    mediated by alpha 4 integrins are essential in placental and cardiac    development. Development (Cambridge, England). 1995; 121:549-560

SUMMARY OF INVENTION Technical Problem

One purpose of the present invention is to provide a cardiac cellculture material which specifically acts on cardiac cells, and toprovide a cell culture substrate on which a wall surface and/or a bottomsurface of the culture substrate having the wall surface and/or thebottom surface are coated with the cardiac cell culture material. Inaddition, another purpose of the present invention is to provide anartificial organ material obtained by culturing a cardiac cell by usingthe cardiac cell culture material, and a method for producing the same.

Solution to Problem

It has been made clear that, in cardiac cell culturing, a functionalcardiac tissue is well constructed by using a cardiac cell culturematerial containing VCAM-1 protein. Therefore, the cardiac cell culturematerial is coated on a wall surface and/or a bottom surface of aculture substrate having the wall surface and/or the bottom surface, andcan be used as a cell culture substrate. A cardiac cell cultured byusing the cardiac cell culture material can be used as an artificialorgan material.

Namely, the present invention includes followings.

[1] A cardiac cell culture material comprising VCAM-1 protein.[2] The cardiac cell culture material according to [1], wherein theVCAM-1 protein is a VCAM-1 separated and purified from an animalmaterial, a VCAM-1 recombinant protein, or a cell expressing VCAM-1protein.[3] The cardiac culture material according to [1] or [2] used forculturing to construct a cardiac tissue.[4] The cardiac cell culture material according to [2] or [3], whereinthe cell expressing VCAM-1 protein is a fibroblast expressing VCAM-1protein.[5] The cardiac cell culture material according to [4], wherein thefibroblast is a cardiac-derived fibroblast.[6] The cardiac cell culture material according to [4] or [5], whereinthe fibroblast is an epicardial-derived fibroblast.[7] A cell culture substrate, wherein a wall surface and/or a bottomsurface of the culture substrate having the wall surface and/or thebottom surface are coated with the cardiac cell culture materialaccording to [1]-[6].[8] An artificial organ material obtained by co-culturing a cardiac cellwith the cardiac cell culture material according to [1]-[6].[9] A method of producing an artificial organ material comprising a stepof co-culturing a cardiac cell with the cardiac cell culture materialaccording to [1]-[6].[10] A reagent for screening a cardiac cell culture material containingan anti-VCAM-1 antibody.[11] A cardiac-derived fibroblast expressing VCAM-1 protein.[12] A method of producing the artificial organ material according to[9] further including a step of separating and collecting a culturedcell from a culture substrate.[13] A method of producing the artificial organ material according to[12], wherein the culture substrate is a temperature responsive culturedish, and wherein the separation is performed by temperature change.[14] A method of producing the artificial organ material according to[9] further including the step of agglomerating the co-cultured materialby using scaffold having thickness in a certain extent.[15] A method of producing the artificial organ produced from theartificial organ material according to [9] comprising step of using a 3Dprinter.

Advantageous Effects of Invention

A functional cardiac cell which can be used in a regenerative medicineand an organizational model can be constructed by culturing a cardiaccell by using the cardiac cell culture material of the presentinvention. The cardiac cell culture material can be coated on a wallsurface and/or a bottom surface of a culture substrate having the wallsurface and/or the bottom surface, which can be used as a cell culturesubstrate. Further, a cardiac cell or a cardiac tissue obtained byculturing can be used as an artificial organ material.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A microscopic observation of NCF, ACF and ADF (photographs). (A)Bright field microscope images of each fibroblast. (B-E) RepresentativeFigures of DDR2, vimentin and αSMA expression (Most of the fibroblastswere not expressing calponin, cytokeratin 11 or NG 2).

FIG. 2 Differences in characteristics of mESC derived cardiac cellsheets that were co-cultured with fibroblasts (photographs). (A) Beforeseparated, many cell masses that were autonomously beating were observedon NCF and ACF co-culture sheet. After decrease in temperature, cellsheet formation was not observed in mESC derived cardiomyocytes andfibroblasts (−). (B) Extracellular action potentials on each of the cellsheets. Action potentials in ACF or NCF co-culture sheet were observedin each channel. However, the action potentials occurred on a one-offbasis on the ADF co-culture sheet (encircling lines indicate the shapesof the cell sheets). (C) Immunofluorescent stain in each of the cellculture dishes which were observed by a confocal microscope. YFP emittedgreen (yellow) fluorescence (YFP: excitation wavelength 514 nm,fluorescence wavelength 527 nm), and vimentin emitted red fluorescence(cy3: excitation wavelength 512 nm, fluorescence wavelength 552 nm), andthe nucleus was stained in hoechst 33258 (blue) (hoechst 33258:excitation wavelength 352 nm, fluorescence wavelength 461 nm). Theconfocal microscopy observation suggested that, the cells co-culturedwith NCF or ACF have a large number of YFP (+) cells, compared with thecells co-cultured with fibroblasts (−) or ADF. (D) Immunofluorescentstain in each of the cell culture dishes observed by a confocalmicroscopy. cTnT was stained by Cy5 (Cy5: excitation wavelength 650 nm,fluorescence wavelength 530 nm), vimentin emitted red fluorescence (cy3:excitation wavelength 512 nm, fluorescence wavelength 552 nm), and thenucleus was stained in hoechst 33258 (blue) (hoechst 33258: excitationwavelength 352 nm, fluorescence wavelength 461 nm). The confocalmicroscopy observation suggested that, the cells co-cultured with NCF orACF have a large number of cTnT (+) cells, compared with the cellsco-cultured with fibroblasts (−) or ADF. (E) The bar graphs showincrease in the numbers of YFP (+) cells or of cTnT (+) cells in each ofthe cell culture dishes. The numbers of YFP (+) cells or of cTnT (+)cells in fibroblasts (−) were set to 1. More numbers of YFP (+) cellsand cTnT (+) cells were observed in NCF or ACF culture dish comparedwith those in the culture dish of ADF co-culture or fibroblasts (−). Inaddition, there is no significant relationship in the number ofcardiomyocytes between NCF and ACF. (N=3, **P<0.01)

FIG. 3 The number of cardiomyocytes at day 1 and day 5 from the cellculture start in each of the cell culture dishes (Photographs). (A)Immunofluorescent stain at day 1 from culture start in each of the cellculture dishes which were used in a confocal microscope. YFP emittedgreen (yellow) fluorescence (YFP: excitation wavelength 514 nm,fluorescence wavelength 527 nm), and cTnT emitted red fluorescence (cy3:excitation wavelength 512 nm, fluorescence wavelength 552 nm), and thenucleus was stained in hoechst 33258 (blue) (hoechst 33258: excitationwavelength 352 nm, fluorescence wavelength 461 nm). (B)Immunofluorescent stain at day 5 from culture start in each of the cellculture dishes which were observed by a confocal microscope. YFP emittedgreen (yellow) fluorescence (YFP: excitation wavelength 514 nm,fluorescence wavelength 527 nm), and cTnT emitted red fluorescence (cy3:excitation wavelength 512 nm, fluorescence wavelength 552 nm), and thenucleus was stained in hoechst 33258 (blue) (hoechst 33258: excitationwavelength 352 nm, fluorescence wavelength 461 nm). (c) The number ofcardiomyocytes in each of the cell culture dishes. The bar graphs showincrease in the numbers of YFP (+) cells and of cTnT (+) cells (Thevalues at day 1 in fibroblasts (−) were set to 1). In the ACF and NCFculture dishes, more numbers of cardiomyocytes were observed at day 5from culture start compared with those at day 1. However, in the otherculture dishes, there was no difference in the number of cardiomyocytesbetween day 1 and day 5. No significant difference was observed betweenACF and NCF. (N=3, **P<0.01)

FIG. 4 Evaluation of proliferation in cardiomyocytes byimmunofluorescent stain (photographs). (A) Immunofluorescent stainobservation of Ki67 positive cardiomyocytes in each of co-culture dishesby using the confocal microscope. cTnT was stained by Cy5 (Cy5:excitation wavelength 650 nm, fluorescence wavelength 530 nm), and Ki67emitted red fluorescence (cy3: excitation wavelength 512 nm,fluorescence wavelength 552 nm), and the nucleus was stained in hoechst33258 (blue) (hoechst 33258: excitation wavelength 352 nm, fluorescencewavelength 461 nm). (B) Percentage of Ki67 (+) or phosphorylated histone3 (phosphor S10; Phh3) (+) cardiomyocytes in each of the culture dishes(N=4, **P<0.01). (C) Immunofluorescence stain observation ofphosphorylated histone 3 (phosphor S10; Phh3) positive cardiomyocytes ineach of the culture dishes by using the confocal microscope. cTnT wasstained by Cy5 (Cy5: excitation wavelength 650 nm, fluorescencewavelength 530 nm), and phosphorylated histone 3 (phosphor S10; Phh3)emitted red fluorescence (cy3: excitation wavelength 512 nm,fluorescence wavelength 552 nm), and the nucleus was stained in hoechst33258 (blue) (hoechst 33258: excitation wavelength 352 nm, fluorescencewavelength 461 nm). (D) Percentage of phosphorylated histone 3 (phosphorS10; Phh3) (+) cardiomyocytes in each of the culture dishes (N=4,**P<0.01). (E) (F) BrdU FACS assay of cardiomyocytes in each of theculture dishes (N=3, **P<0.01). (G) Immunofluorescence stain observationof YFP (+) and of cTnT (+) at day 1 and day 5 from culture start in theinsert culture dishes by using the confocal microscope. YFP emittedgreen (yellow) fluorescence (YFP: excitation wavelength 514 nm,fluorescence wavelength 527 nm), and cTnT emitted red fluorescence (cy3:excitation wavelength 512 nm, fluorescence wavelength 552 nm), and thenucleus was stained in hoechst 33258 (blue) (hoechst 33258: excitationwavelength 352 nm, fluorescence wavelength 461 nm). (H) The bar graphsshow increase in the numbers of YFP (+) cells and of cTnT (+) cells atday 1 and at day 5. The numbers of YFP (+) cells and of cTnT (+) cellsat day 1 were set to 1. The proliferation of cardiomyocytes was observedat day 5 (N=4, **P<0.01).

FIG. 5 (A) Comprehensive gene cluster analysis of ADF and NCF(photograph). This gene heat map shows a remarkable difference betweenADF and NCF. This map was divided into two groups. The first groupconsisted of only ADF, and the second group consisted of only NCF. (B)The VCAM-1 gene expression level was examined by real time PCR. TheVCAM-1 expression level was significantly high in NCF. The number ofVCAM-1 genes in NCF was 16 times higher than that in ADF (N=3, *P<0.05).(C-D) The expression level of VCAM-1 protein in NCF and ADF in westernblot analysis. The following transient overexpression cell lysate wasused as a positive control: Sol8 (SantaCruz, Calif., USA). The labelpeak of β-actin of each cell was set to 1 (N=3, **P<0.01). (E)Immunofluorescence stain of the VCAM-1 receptor (α4β1) on mESC derivedcardiomyocytes. (F) Western blot analysis of the VCAM-1 receptor on mESCderived cardiomyocytes. The following transient overexpression celllysate was used as a positive control: Jurkat whole cell lysate.

FIG. 6 Identification of cardiac growth factor by immunofluorescencestain analysis (photographs). (A-B) Immunofluorescence stain observationof the effect of neutralizing antibodies on cardiomyocytes at day 5. YFPemitted green (yellow) fluorescence (YFP: excitation wavelength 514 nm,fluorescence wavelength 527 nm), and cTnT emitted red fluorescence (cy3:excitation wavelength 512 nm, fluorescence wavelength 552 nm), and thenucleus was stained in hoechst 33258 (blue) (hoechst 33258: excitationwavelength 352 nm, fluorescence wavelength 461 nm). When NCF andcardiomyocytes were co-cultured by using a VCAM-1 neutralizing antibody,the number of cardiomyocytes was decreased at day 5. Meanwhile, when anisotype control was used, there was no effect on the number ofcardiomyocytes at day 5. (N=3, **P<0.01). (C-D) Immunofluorescence stainobservation of the effect of VCAM-1 soluble protein on cardiomyocytes atday 5. YFP emitted green (yellow) fluorescence (YFP: excitationwavelength 514 nm, fluorescence wavelength 527 nm), and cTnT emitted redfluorescence (cy3: excitation wavelength 512 nm, fluorescence wavelength552 nm), and the nucleus was stained in hoechst 33258 (blue) (hoechst33258: excitation wavelength 352 nm, fluorescence wavelength 461 nm).Cardiomyocyte growth effect was obtained by culturing with VCAM-1soluble protein (10 μg/mL). Moreover, the number of cardiomyocytes onDay 5 was comparable to that in co-culture with NCFs. (N=3, *P<0.05,**P<0.01).

FIG. 7 The results of FACS analysis of cardiac fibroblasts derived fromneonatal mice. (A-C) The results of staining with an anti-VCAM-1antibody are shown. (D) The result of a negative control by stainingdermal fibroblasts only with a secondary antibody is shown.

FIG. 8 The optimum compounding concentration of VCAM-1 (+) cardiacfibroblasts. (A) Phase difference images of VCFs (VCAM-1 (+)) and VNCFs(VCAM-1 (−)) isolated by a magnetic cell separator (MACS). (B)Fluorescence images (photographs) of VCFs (VCAM-1 (+)) and VNCFs (VCAM-1(−)) isolated by a magnetic cell separator (MACS). VCAM-1 emits redfluorescence, and Hoechst 33258 emits blue fluorescence. Scale bar=200μm.

FIG. 9 Evaluation of optimum compounding ratio of VCFs. (A) Fluorescenceimages (photographs) showing the results of co-culturing cardiomyocytesexpressing GFP with ADFs, NCFs, or VCFs and/or VNCFs. ADFs=mouseadult-derived dermal fibroblasts, VCFs=VCAM-1 positive mouse neonatalcardiac fibroblasts, VNCFs=VCAM-1 negative mouse neonatal cardiacfibroblasts, and NCFs=neonatal mouse cardiac fibroblasts. GFPcardiomyocytes emit green fluorescence, and Ki 67 emits redfluorescence, and cTnT was stained by Cy5 (Cy5: excitation wavelength650 nm, fluorescence wavelength 530 nm). Magnification is ×20. (B) A bargraph showing the results of co-culturing cardiomyocytes expressing GFPwith ADFs, NCFs, or VCFs and/or VNCFs. The numbers of GFP (+) cells andcTnT (+) cells were set to 1 when cardiomyocytes and ADFs wereco-cultured at the concentrations of 80% and 20%, respectively. N=4,**P<0.01.

FIG. 10 Evaluation of localization (N=4) of cardiomyocytes andfibroblasts at day 5 in a tissue created at the concentration of 80%cardiomyocytes and 20% VCFs.

FIG. 11 Evaluation of division (proliferation) effect, migration effectand ability of constructing a network of cardiomyocytes for 5 days ofculturing under the condition of co-culturing with NCFs and ADFs(photographs). YFP expressing cardiomyocytes emit green fluorescence,and DsRed fibroblasts emit red fluorescence. Magnification is ×200.

FIG. 12 A shows fluorescence images indicating division (proliferation)of cardiomyocytes for three days of culturing at the time whenco-culturing ES-derived cardiomyocytes with VCFs (VCAM-1 (+)) or VNCFs(VCAM-1 (−)) (photographs). GFP expressing cardiomyocytes emit greenfluorescence. Magnification is ×100. B shows the results evaluating themigratory ability by calculating the total migratory distance for 3 daysof culturing at the time when co-culturing ES-derived cardiomyocytes,and VCFs or VNCFs. N=5, *P<0.05.

FIG. 13 The results of analyzing the localization of PlateletEndothelial Cell Adhesion Molecule-1 (PECAM-1, CD31) and VCAM-1 positivecardiac fibroblasts with flow cytometry.

FIG. 14 (A) Localization of VCFs in a biological heart (N=3). (B)Localization of VCFs in cardiac fibroblasts (N=3). (C) Localization ofCD31-positive fibroblasts in a biological heart (N=3). (D) Localizationof CD31 positive fibroblasts in cardiac fibroblasts (N=3).

DESCRIPTION OF EMBODIMENTS

The present embodiment relates to a cardiac cell culture materialcontaining VCAM-1. In the present embodiments, the “cardiac cell culturematerial” may be any material that is used when culturing a cardiaccell. For example, the material includes but is not limited to a reagentsuch as a protein, and a peptide, etc. to be added to a culture medium,and a material, etc. for coating a bottom surface or a wall surface of aculture substrate of a culture vessel, etc. such as a petri dish and aflask, and the like. Examples of these cell culture substrate, in whicha wall surface and/or a bottom surface of the culture substrate havingthe wall surface and/or the bottom surface are coated with the cardiaccell culture material include microcarrier, and cell culture bag, etc.

VCAM-1 (vascular cell adhesion molecule-1) is a known protein as a celladhesion molecule that expresses in a vascular endothelial cell, and thelike. For example, in the case of humans, VCAM-1 includes but notlimited to a protein encoded by a gene described in accession numberNM_001078, etc. of NCBI (National Center for Biotechnology Information),and also includes an isoform obtained by alternative splicing. TheVCAM-1 protein in the present embodiment includes VCAM-1 which isexpressing on a cell surface, a soluble VCAM-1, various mutants one or aplurality of, for example, 1-20, 1-15, 1-10 or 1-5 of amino acids ofwhich have been deleted from, substituted from, or added to an aminoacid of VCAM-1 protein and having the same activity as VCAM-1 protein. AVCAM-1 protein in an animal material which has been separated andpurified by a well-known method and a recombinant protein may be used asthe VCAM-1 in the present embodiment. For example, the animal materialincludes but is not limited to humans; experimental animals such asmice, rats, guinea pigs, hamsters, pigs, monkeys and rabbits; andbacteria such as E. coli, etc. A commercially available recombinantprotein may be also used.

Moreover, a cell that is expressing VCAM-1 may be used as VCAM-1 of thepresent embodiment. In order to screen a cell that is expressing VCAM-1,a publicly known cell sorting method may be used. For example, the cellsorting method includes but not limited to flow cytometry using ananti-VCAM-1 antibody, magnetic bead method, affinity column method, andpanning method.

Anti-VCAM-1 antibodies are not particularly limited. Commerciallyavailable anti-VCAM-1 antibodies may be used, and a product produced bya known method by using VCAM-1 as an antigen may be also used. Moreover,as far as the cells that are expressing VCAM-1 may be screened, eithermonoclonal antibody or polyclonal antibody may be used; however, it ispreferred to use monoclonal antibody from the viewpoint of specificity.

Namely, the methods of screening the cardiac cell culture materials ofthe present embodiment include, a step of preparing cells, a step ofperforming cell sorting to the cells by using a VCAM-1 antibody, and astep of collecting only cells that have been judged to be expressingVCAM-1 as a result of the cell sorting.

As the cell that is expressing VCAM-1, the types are not limited as faras VCAM-1 is expressed. However, it is preferred to use fibroblasts. Thefibroblasts include all the cells that will ultimately becomefibroblasts or myofibroblasts. Namely, the scope of fibroblasts of thepresent embodiment includes the cells that are in the middle ofdifferentiation or a maturation stage and cannot be identified asfibroblasts or myofibroblasts at that time as far as the cells willultimately become fibroblasts or myofibroblasts. Moreover, the scope offibroblasts of the present embodiment includes the cells that are notcalled as fibroblast, such as stromal cells, interstitial cells,progenitor cells, precursor cells, stem cells, or the like, as far asthe cells have the same functions and activities as fibroblasts andexpress VCAM-1.

Derivation of fibroblasts is not limited. Pluripotent stem cells such asES cells, iPS cells and muse cells, and adult stem cells such asmesenchymal stem cells may be differentiated and used, and primary cellstaken from animals may be used, and established cells may be used.However, cardiac-derived fibroblasts are preferably used, and amongthem, epicardium-derived fibroblasts are in particular preferred to beused. In a case where established cells are used, processing of cellsorting may be omitted by selecting the cells that are known to expressVCAM-1. The animals from which fibroblasts are derived may beappropriately selected in accordance with the animals from which thecells to be co-cultured are derived. The animals, for example, includehumans; experimental animals such as mice, rats, guinea pigs, hamsters,pigs, monkeys and rabbits; pet animals such as dogs, cats and birds; andlivestock such as cattle, horses, sheep and goats. In a case wherefibroblasts are taken from animals, the fibroblasts may be of at anytime of the animals such as fetus, neonate, infant, adult, and there isno limit.

The cardiac cell culture material of the present embodiment may be acomposition containing physiological saline, cell culture solution, orcell preservation solution, etc. for maintenance or preservation ofVCAM-1 protein or cells that are expressing VCAM-1 protein. There is nolimit on the contents contained in the composition as far as thecontents do not impair the function of VCAM-1. Moreover, the state ofthe cardiac cell culture material of the present embodiment may beliquid, gel-like, freezed, or freeze-dried, and the state thereof is notlimited.

Further, the cardiac cell culture material may include fibroblastsregardless of presence or absence of VCAM-1 protein. The fibroblastsinclude all the cells that will ultimately become fibroblasts ormyofibroblasts. Namely, even if the cells are in the middle ofdifferentiation or a maturation stage and cannot be identified asfibroblasts or myofibroblasts at that time, if the cells are those thatwill ultimately become fibroblasts or myofibroblasts, the cells may beused without limit. Among them, fibroblasts that are expressing CD31(vascular endothelial cell marker) are preferred. When fibroblasts thatare expressing VCAM-1 protein are used as VCAM-1 protein, the ratio ofVCAM-1 protein expressing cells (cell number):CD31 expressing cells(cell number) are preferably 5:5-9:1, more preferably 5:5-8.2, even morepreferably 6:4-8.2, and may be 7:3-8:2.

The present embodiment relates to an artificial organ material in whichthe ratio of cardiac fibroblasts expressing VCAM-1 to all the cells is50% or higher. The ratio of the cardiac fibroblasts is 50% or higher,and preferably 60% or higher, and more preferably 70% or higher, andeven more preferably 80% or higher, and most preferably 90% or higher.The artificial organ material may be obtained by co-culturing cardiacfibroblasts that express VCAM-1 with cardiomyocytes, and the ratio ofthe cardiac fibroblasts at the start of co-culturing is normally 3% orhigher, preferably 4% or higher, more preferably 6% or higher, and evenmore preferably 8% or higher, and most preferably 9% or higher.Meanwhile, the ratio of the cardiac fibroblasts at the start ofco-culturing is preferably 30% or lower, preferably 20% or lower, andmost preferably 20%. The present invention also relates to an artificialorgan material obtained by culturing a cardiac cell with theabove-mentioned cardiac cell culture material, or a method for producingthe same. Namely, the cardiac cell culture material of the presentembodiment can construct a functional cardiac tissue that can be used ina regenerative medicine and organizational model by culturing with acardiac cell. The cardiac tissue can be used as an artificial organmaterial. The artificial organ material can be of any form. For example,it can be adhered to a damaged part of organs such as heart in the formof sheet. Therefore, one embodiment is a method for curing heartdiseases by attaching the sheet to a damaged part of the heart. Theartificial organ material can be also transplanted to a defect site ofan organ after it is laminated or it is agglomerated by using scaffold,which has thickness in a certain extent. The material of the scaffoldincludes but not limited to hydroxyapatite, atelocollagen, and gel.Further, the artificial organ material can be used for celltransplantation, academic research, etc. as it is in the state of theculture cell, without making it to be a particular form. Furthermore, anartificial organ can be produced from the artificial organ material byusing a 3D printer. The produced artificial organ not only can be usedfor transplantation but also can be widely used for safety pharmacologytest and preclinical research, etc.

In the present embodiment, “constructing a cardiac tissue” meansconstructing a tissue having at least one of the cardiac functions suchas promoting division of cardiac cells, and providing uniform beatingthroughout a whole tissue, which can be used for regenerative medicineand a tissue model. The cardiac functions include all the known cardiacfunctions such as autonomous pulsating ability, contraction andrelaxation ability, impulse conduction ability, and hormone secretionability, etc. The cardiac functions are not limited to functions whichonly the heart has. For example, a muscle cell also has the contractionand relaxation ability. However, even if other cells have an equivalentfunction, it does not affect the definition of the cardiac functions ofthe present embodiment. Further, with respect to the cardiac functions,there is no limit on highness and lowness of the functions as long asthey are suitable for use purpose of a cardiac tissue. For example, forthe purpose of producing an artificial heart, it is required to have acontraction and relaxation ability to the extent that it can pump outblood thought out the body; however, for the purpose of academicresearch, etc. of contraction and relaxation ability in vitro, it issatisfied if contraction and relaxation ability is detected by somemeans.

In the artificial organ material, or the method to produce the same ofthe present embodiment, cardiac cells to be used include all the cellsthat constitute the heart such as cardiomyocytes, smooth muscle cells,pacemaker cells and vascular endothelial cells. The derivation of thecardiac cells can be appropriately set in accordance with the purpose ofuse as an artificial organ material. For example, for the purpose oftransplantation to humans, human-derived cardiac cells may be used, andfor the purpose of constructing a tissue model in a mouse experiment,mouse-derived cardiac cells may be used. Furthermore, a cardiac cell ofany period from fetus, newborn, pediatric and adult can be used, andthere is no limit on the period. The cardiac cell of the presentembodiment is preferred to be produced from pluripotent stem cells suchas ES cells, iPS cells, and muse cells, and adult stem cells such asmesenchymal stem cells.

The “culturing” of the present embodiment can be carried out by apublicly known cell culturing method, and there is no limit on thecondition of the culturing as long as a cardiac cell culture materialand a cardiac cell are present in a culture vessel, or are immersed inthe same culture medium. In a case where the cardiac cell culturematerial are cells which are expressing VCAM-1 protein, the mixingpercentage of the cells (cell number) that are expressing VCAM-1 tocardiac cells are preferably 3-20%, more preferably 6-18% and mostpreferably 9-16%.

In the present embodiment, a culture liquid used for the culturing canbe appropriately set in accordance with a kind of cell to be cultured.For example, DMEM, α-MEM, RPMI-1640, and the like may be used.Nutritional substances such as FCS and FBS and antibiotics may be addedto the culture liquid. Growth factor and cytokines such as fibroblastgrowth factors (FGF) may also be added to the culture liquid.

With respect to the cultivation period, the number of days until thedesired cell number and/or function are obtained may be appropriatelyset. For example, the periods include 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, 14 days, 1 month, 2 months, 3 months, 4 months, 5 months, and 6months. The cultivation temperature may be appropriately set inaccordance with the kinds of cells to be cultured. For example, thetemperature may be 10-60° C., preferably 20-50° C., and more preferably30-40° C.

The production method of the present embodiment may further include thestep of collecting cultured cells. The “cultured cells” may include bothfibroblasts and cardiac cells, and may only include the cardiac cells.With respect to the step to collect a cell, the cell may be separatedand collected by using proteases such as trypsin. However, it ispreferred that cell is separated and collected by the change intemperature by using a temperature responsive culture dish capable ofseparating a cell while retaining an extracellular matrix, etc.

EXAMPLES

The present invention is further described below in detail withreference to the following examples; however, it should be construedthat the invention is no way limited to those examples.

Example 1 Materials and Methods <Animals and Reagents>

Wild-type C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan).B6 Cg-Tg (CAG-DsRed*MST) 1Nagy/J mice were purchased from The JacksonLaboratory (Bar Harbor, Me.). All the experimental protocols wereapproved by the Institutional Animal Care and Use Committee of TokyoWomen's Medical University. The following antibodies were used forimmune cytochemistry, western blot and flow cytometric analysis (FACS):rabbit polyclonal anti-discoidindomein receptor tyrosine kinase 2 (DDR2)(GeneTex, Irvine, Calif.); guinea pig monoclonal anti-vimentin (Progen,Heidelberg, Germany); mouse monoclonal anti-NG2 (Millipore, Temecula,Calif.); Rabbit polyclonal anti-alpha smooth muscle actin (Abcam,Cambridge, UK); mouse monoclonal anti-cardiac troponin T (cTnT) (ThermoScientific, Rockford, Ill.); mouse monoclonal anti-cytokeratin11 (EXBIO,NadSafinou, CZ); rabbit polyclonal anti-Ki67 (Abcam, Cambridge, UK);rabbit polyclonal anti-Histon H3 (phosphor S10) (Abcam, Cambridge, UK);rat monoclonal anti-integrin α4/β1 (Abcam, Cambridge, UK); recombinantmouse VCAM-1/CD106 Fc chimera (R&D systems, Minneapolis, Minn.). Unlessotherwise specified, all reagents were purchased from Sigma-Aldrich.Secondary antibodies were purchased from Jackson ImmunoResearchLaboratories (West Grove, Pa.).

<Mouse ES Cell Cultures>

The maintenance of mESC expressing the neomycin phosphotransferase geneunder the control of the α-myosin heavy chain promoter and cardiomyocytedifferentiation and purification were described previous report(Matsuura K, et al. Biomaterials. 2011; 32:7355-7362). Briefly, forcardiac induction and cardiomyocyte purification, trypsinized ES cellswere seeded at 5×10⁴ cells/mL (total, 125 mL/flask) into spinner flasks(Integra Biosciences, Zizers, Switzerland) and cultured with DMEMsupplemented with 10% FBS for 10 days, then these differentiated cellswere treated with neomycin for further 8 days.

<Fibroblast Isolations>

Fibroblasts were obtained from Wild-type C57BL/6 mice (Neonatal, 1 day;Adult, 10-12 weeks).

Neonatal cardiac fibroblasts (NCFs) were obtained from hearts ofneonatal mice (1 day of age) as described previous report (Matsuura K,et al, Biomaterials. 2011; 32: 7355-7362). NCFs from passage 3 were usedfor the experiments.

Adult cardiac fibroblasts (ACFs) were obtained from hearts of adult mice(10-12 weeks) using the explant culture method as follow. First heartswere washed with PBS (−) and cut into circa 5 mm² species. These specieswere covered with sterilized cover glasses and cultured with DMEMsupplemented with 10% FBS on 10 cm culture dishes. 2 weeks afterstarting culture, cells were dissociated with 0.25% Trypsin/EDTA andsubcultured to other 10 cm dishes. ACFs from passage 3 were used for theexperiments.

Adult dermal fibroblasts (ADFs) were obtained from dorsal dermal tissueof adult mice (10-12 weeks). First harvested dermal tissues were treatedwith Dispase I [1000 U/mL] (Eidea inc.) over night at 4° C. Next, thetissues were cut into circa 1 mm² species. These species were coveredwith sterilized cover glasses and cultured with DMEM supplemented with10% FBS on 10 cm culture dishes. 2 weeks after starting culture, cellswere dissociated with 0.25% Trypsin/EDTA and subcultured to another 10cm dishes. ADFs from passage 3 were used for the experiments.

In some experiments, NCFs and ADFs were isolated from B6.Cg-Tg(CAG-DsRed*MST) 1Nagy/J mice (Neonatal: 1 day, Adult: 10 weeks) with thesame methods as described above.

<Cell Sheet Preparation>

Before seeding cells, the surface of temperature-responsive culturedishes (UpCell; CellSeed inc.) was coated with FBS for 2 h. mESC-derivedcardiomyocytes were co-cultured with each type of fibroblasts at theratio of 8:2 with DMEM supplemented with 10% FBS (3.2×10⁵ cells/cm²).After 5 days of culture, the cells were incubated at 20° C. fordetaching cell sheets. Bright field images of samples were obtained by aNikon ECLIPSE Ti (Nikon, Tokyo, Japan).

<Electrophysiological Analysis>

The electrical activities of the cardiomyocyte sheets were obtained fromthe extracellular potentials measured by a multi-electrode array (MED)system (Alpha MED Sciences inc.) as described previous report (MatsuuraK, et al, Biomaterials. 2011; 32:7355-7362).

<Immunocytochemistry>

Cells were fixed with 4% paraformaldehyde and subjected toimmunostaining as described previous report (Matsuura K, et al,Biomaterials. 2011; 32:7355-7362). Images of the stained samples wereobtained by an ImageXpress Ultra Confocal High Content Screening System(Molecular Devices, CA, USA). Image analysis data was obtained by aMetaExpress software (Molecular Devices inc.).

<FACS Analysis>

Incubating cells (5×10⁵ cells) were stained with BrdU at a finalconcentration of 10 μM in cell culture medium. BrdU staining for a FACSanalysis was performed as described in a BrdU Flow Kits InstructionManual (BD Pharmingen, Franklin Lakes, N.J.). Briefly, cells were fixedand permeabilized with BD Cytofix/Cytoperm Buffer, then exposedincorporated BrdU with DNase. BrdU staining was performed withAPC-anti-BrdU antibody (BD Pharmingen, Franklin Lakes, N.J.). Sampleswere analysed with a Gallios (Beckman Coulter, Brea, Calif.). Thefollowing reagents were used for the analysis: BD Cytofix/CytopermBuffer (BD Pharmingen, Franklin Lakes, N.J.); BD Perm/Wash Buffer (10×)(BD Pharmingen, Franklin Lakes, N.J.); BD Cytoperm Plus Buffer (10×) (BDPharmingen, Franklin Lakes, N.J.); BrdU (10 mg/mL) (BD Pharmingen,Franklin Lakes, N.J.); DNase (BD Pharmingen, Franklin Lakes, N.J.).

<Time-Laps Photography>

Samples were observed five days in 5% CO₂ at 37° C. with a BZ-9000Fluorescence Microscope (Keyence, Osaka, Japan).

<RNA Extraction and Comprehensive Genetic Analysis>

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad,Calif.) according to the manufacturer's instructions. Total RNA wasfurther purified using the Qiagen RNeasy Mini Kit (QIAGEN, Valencia,Calif.) according to the manufacturer's instructions.

RNA quantity and quality were determined using a Nanodrop ND-1000spectrophotometer (Thermo Fisher Scientific Inc., Waltham, Mass.) and anAgilent Bioanalyzer (Agilent Technologies, Palo Alto, Calif.), asrecommended.

For cRNA amplification and labeling, total RNA was amplified and labeledwith Cyanine 3 (Cy3) using Agilent Low Input Quick Amp Labeling Kit,one-color (Agilent Technologies, Palo Alto, Calif.) following themanufacturer's instructions. Briefly, 100 ng of total RNA was reversedtranscribed to double-strand cDNA using a poly dT-T7 promoter primer.Primer, template RNA and quality-control transcripts of knownconcentration and quality were first denatured at 65° C. for 10 min andincubated for 2 hours at 40° C. with 5× first strand Buffer, 0.1 M DTT,10 mM dNTP mix, and Affinity Script RNase Block Mix. The AffinityScriptenzyme was inactivated at 70° C. for 15 min.

cDNA products were then used as templates for in vitro transcription togenerate fluorescent cRNA. cDNA products were mixed with a transcriptionmaster mix in the presence of T7 RNA polymerase and Cy3 labeled-CTP andincubated at 40° C. for 2 hours. Labeled cRNAs were purified usingQIAGEN's RNeasy mini spin columns and eluted in 30 μl of nuclease-freewater. After amplification and labeling, cRNA quantity and cyanineincorporation were determined using a Nanodrop ND-1000 spectrophotometerand an Agilent Bioanalyzer.

For Sample hybridization, 1.65 μg of Cy3 labeled cRNA were fragmented,and hybridized at 65° C. for 17 hours to an Agilent Mouse GE 4x44Kv2Microarray (Design ID: 026655). After washing, microarrays were scannedusing an Agilent DNA microarray scanner.

For data analysis of microarray, intensity values of each scannedfeature were quantified using Agilent feature extraction softwareversion 10.7.3.1, which performs background subtractions.

We only used features that were flagged as no errors (present flags) andexcluded features that were not positive, not significant, not uniform,not above background, saturated, and population outliers (marginal andabsent flags). Normalization was performed using Agilent GeneSpring GXversion 11.0.2. (per chip: normalization to 75 percentile shift; pergene: normalization to median of all samples). There are total of 39,429probes on Agilent Mouse GE 4x44Kv2 Microarray (Design ID: 026655)without control probes.

The altered transcripts were quantified using the comparative method. Weapplied 2-fold change in signal intensity to identify the significantdifferences of gene expression in this study.

<Quantitative Real-Time PCR Analysis>

Complementary DNA was generated from total RNA with High Capacity cDNAReverse Transcription Kit (Applied biosystems). As the PCR-relatedprimers, VCAM-1 Gene Express Assays (life Technology) was used.

Each RT-PCR included 10 minutes at 25° C., 120 minutes at 37° C., and 5seconds at 85° C. with iCycler (BIO-RAD). cDNA template (1 μg) was usedfrom each sample. TaqMan probe real-time PCR studies were performed withTaqMan Gene Expression Assays (Applied biosystems). All experiments wereconducted in triplicate. Samples were cycled 40 times with an 7300 RealTime PCR System (Applied Biosystems) as follows: 2 minutes at 50° C. and10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and1 minute at 60° C. Relative quantification was calculated according tothe ΔΔCT method for quantitative real-time PCR using a Gap DH gene asendogenous control.

<Western Blotting>

NCFs or ADFs were lysed in Laemmli sample buffer (BIO-RAD, CA, USA),protease inhibitor (Boehringer Mannheim, Indianapolis, Ind.) and2-mercaptoethanol (Wako Pure Chemical Industries, Japan). The sampleswere separated on a 4% to 12% Bis-Tris Gels (Life Technologies, MD,United States), electrotransferred to a iBlot Transfer Stack,nitrocellulose, regular-size (Life technologies, MD, United States) withiBlot 7-Minute Blotting System (Life technologies, MD, United States),and processed for chemiluminescence analysis with Amersham ECL PrimeWestern Blotting Detection Reagent (GE Healthcare, PA, United States).Band intensity was analyzed using LAS4000 (Fujifilm, Tokyo, Japan) andNIH image software (version 1.46r). The following cell transientoverexpression lysates were used for positive controls: K562 (Humanerythromyeloblastoid leukemia cell line) for Col11a1 (Abcam, CB, UK);Sol8 (SantaCruz, Calif., USA) for Vcam-1; ITGB1 293T for β1/CD29(Abnova, Taipei, Taiwan); Jurkat Whole Cell Lysate for integrin α4β1(SantaCruz, Calif., USA).

<Neutralizing Antibodies Assay>

The following antibodies and culture dishes were used for neutralizingantibody assay: anti-VCAM-1 (LifeSpan Biosciences, Seattle, Wash.); goatIgG isotype control (LifeSpan Biosciences, Seattle, Wash.). Cell CultureInserts for 24-well plates. 0.4 μm pores, Translucent, High Density PETMembrane (BD Pharmingen, Franklin Lakes, N.J.).

After the pretreatment with the antibodies at 10 μg/mL for 30 min,fibroblasts were seeded onto the upper layer of insert culture dishes(2.4×10⁵ cells). mESC-derived cardiomyocytes were seeded onto the belowlayer (4.8×10⁵ cells). The culture medium with the antibody at 10 μg/mLwas changed every day until 5 days.

<Statistical Analysis>

All data were presented as the mean±SD. The significance of thevariation among different groups was determined by One-Way ANOVAAnalysis. And then, the difference between two groups was determined byTukey-Kramer Multiple Comparison Test using Statcel Software. pvalue<0.05 was considered to be significantly different.

2. Results of Experiments

<Cell Sheet Creation Using mESC-Derived Cardiomyocytes and Fibroblast>

At first we evaluated the characterization of cells that we were goingto use for the co-culture experiments. The phase contrast imagesrevealed that cells isolated from neonatal hearts, adult hearts andadult dermal tissue showed the fibroblast-like morphology (See FIG. 1A).Since there are not specific antibodies for fibroblasts, we tried toexamine the expression of the proteins that are known to be expressed infibroblasts such as DDR2 (CD167b), vimentin and αSMA. As shown in FIGS.1B to 1E, almost all of each type of cells expressed DDR2, vimentin andαSMA, but not Calponin (smooth muscle cell marker), Cytokeratin(epithelial cell marker) and NG2 (pericyte marker). On the basis ofthese findings, we used these cells as fibroblasts followingexperiments.

According to our previous findings that certain extent amounts offibroblasts were necessary for fabricating cell sheet using mESC-derivedcardiomyocytes and the optimal ratio of cardiomyocytes/fibroblasts was8:2 (Biomaterials. 2011:32:7355-7362), we tried to create cardiac cellsheets using mESC-derived cardiomyocytes and 3 types of fibroblasts(ACFs, ADFs and NCFs) on UpCell temperature-responsive culture dishes.When cardiomyocytes were co-cultured with ACFs or NCFs, beatingcardiomyocytes were equally distributed all over the area. Conversely,when cardiomyocytes were co-cultured with ADFs, nearby beating cellswere aggregated. After 5 days cultivation, when the cultivationcondition were changed from 37° C. to 20° C., monolayered cell sheetswere created in every condition with fibroblasts, but not in thecondition without fibroblasts (FIG. 2A).

Next we examined the electrophysiological evaluation of the cell sheetsusing a MED system (Biomaterials. 2011; 32:7355-7362, Biomaterials.2006; 27:4765-4774). Consistent with the microscopical observation (FIG.2A), the extracellular action potential was observed at each channel incell sheets with ACFs and NCFs (FIG. 2B). Since it was recognized that,in these cell sheets, the entire sheets were uniformly beating, it wassuggested that an electronic network was fabricated in the sheets andthese cell sheets can carry out electric propagation. Meanwhile, in thecell sheets co-cultured with ADFs, the extracellular action potentialwas only observed in limited areas.

To confirm the difference of cardiomyocytes distribution among cellsheets, cofocal microscopic analysis was performed. As shown in FIGS. 2Cto 2E, the number of YFP (+) cells and cardiac troponin T (cTnT) (+)cells, indicatives of mESC-derived cells, in cell sheets with ACFs andNCFs were more than those in cell sheets with ADFs. The number ofcardiomyocytes in cell sheets with ADFs was comparable to that incondition without fibroblasts. In addition, there is no significantcorrelation on the number of cardiomyocytes between cell sheetsco-cultured with ACFs and NCFs. These findings suggest that every kindof fibroblasts was useful for fabricating cell sheet, but fibroblastsderived from hearts might be better for fabricating more functionalcardiac cell sheets.

<Cardiomyocyte Proliferation in Cellsheets>

To investigate the cause of the different number of cardiomyocytesbetween cell sheets co-cultured with heart-derived fibroblasts anddermal tissue-derived fibroblasts, the number of cardiomyocytes wasexamined at day 1 and day 5 in co-culture (FIGS. 3A to C). At day 1, thenumber of cardiomyocytes was identical among conditions, suggesting thateach type of fibroblasts did not affect the initial adherence ofcardiomyocytes after seeding. In co-culture with ACFs and NCFs, thenumber of YFP (+) and cTnT (+) cardiomyocytes at day 5 was significantlyhigher than that at day 1. On the other hand, in co-culture with ADFs orin cardiomyocytes monoculture condition, the number of cardiomyocytes atday 5 was similar to that at day 1. The time-lapse image analysis usingYFP (+) cardiomyocytes and fibroblasts isolated from DsRed mice showedthat cardiomyocytes migrated and proliferated and constructed the mutualnetwork formation in co-culture with NCFs. Conversely in co-culture withADFs, cardiomyocytes showed less proliferation and did not construct thenetwork formation. These findings suggest that fibroblasts from hearts,but not fibroblasts from dermal tissue might induce proliferation ofmESC-derived cardiomyocytes in co-culture condition.

The proliferation of cardiomyocytes among conditions was confirmed bythe immune cytochemical analysis. As shown in FIG. 4A to D, thepercentage of Ki67 (+) cells and phospho histone H3 (PHH3) (+)cardiomyocytes in co-culture with NCFs were significant higher thanthose in co-culture with ADFs and in cardiomyocytes monoculturecondition. Furthermore BrdU incorporation assay also showed thesignificant increase of the percentage of proliferative cardiomyocytesin co-culture with NCFs compared with that in co-culture with ADFs andin cardiomyocytes monoculture condition (FIGS. 4E and F). These findingsstrongly suggest that heart-derived fibroblasts induce proliferation ofcardiomyocytes.

To investigate the underlying mechanisms on the proliferation ofcardiomyocytes in co-culture with NCFs, mESC-derived cardiomyocytes andNCFs were cultured using cell culture inserts. In this experiment, NCFswere cultured on the upper layer and cardiomyocytes were cultured on thelower layer. The number of cardiomyocytes at day 5 was remarkably higherthan that at day 1 in the presence of NCFs (FIG. 4G). However, thedegree of the increase on cardiomyocyte number in the cell cultureinsert experiments between day 1 and day 5 (˜1.8 times) (FIG. 4H) waslower than that in co-culture condition (˜2.5 times). These findingsindicate it might promote the cardiomyocyte proliferation that thesoluble factors secreted from NCFs and the cell-cell interaction betweencardiomyocytes with cardiac fibroblasts.

<Comprehensive Genetic Analysis of NCFs and ADFs>

To identify the factors that are responsible for involved in theseeffects, we performed comprehensive genetic analysis between NCFs andADFs using a microarray analysis. As shown in FIG. 5A, many differencesin gene expression were observed between NCFs and ADFs. Over 500 genesshowed more than 10 times enhanced expression in NCFs compared withADFs. After choosing the cardiovascular-related genes from the lists, 20genes were remained. Furthermore when we selected genes that werereported the embryonic lethal phenotype causing a disorder to generateheart in knock out mouse model and also act as a soluble factor and ansdhesive factor, Vcam-1 was remained. The enhanced expression of Vcam-1in NCFs compared with ADFs was confirmed by quantitative RT-PCR andwestern blot analysis (FIGS. 5B to D).

<VCAM-1-Dependent Cardiomyocyte Proliferation in Co-Culture with CardiacFibroblasts>

Since integrin α4β1 is known to be the principal co-receptor of VCAM-1,we examined the integrin α4β1 expression in mESC-derived cardiomyocytes.As shown in FIGS. 5E and F, almost all of mESC-derived cardiomyocytesshowed generation of integrin α4β1.

Next we elucidated whether VCAM-1 contributed to cardiacfibroblasts-mediated cardiomyocyte proliferation using neutralizingantibodies. After the pretreatment of NCFs with anti-VCAM-1 antibodies,NCFs and mESC-derived cardiomyocytes were cultured using cell cultureinserts. Anti-VCAM-1 antibody treatment significantly inhibited cardiacfibroblast-mediated increase of cardiomyocyte number (FIGS. 6A and B).

Finally we evaluated the direct effects of VCAM-1 on the proliferationof cardiomyocytes. One day after starting culture, cardiomyocytes weretreated with VCAM-1 recombinant protein until day 5. As shown in FIGS.6C and D, VCAM-1 treatment increased the number of cardiomyocytescompared with control. These findings suggest heart-derived fibroblastsmight induce cardiomyocyte proliferation through fibroblasts-mediatedVCAM-1 and integrin α4β1 in cardiomyocytes.

To confirm importance of VCAM-1 positive cells in constructingfunctional cardiac cell sheets, we measured the percentage of VCAM-1positive cells in organism-derived cardiac fibroblasts.

Cardiac fibroblasts were dissected and collected from neonatal mice (1day) of C57/BL6 mice, and skin fibroblasts were dissected and collectedfrom adult mice (10-12 weeks). Each of the fibroblasts wereadhesion-cultured up to passage 3, and the cell volume of 1×10⁷ cellsper condition was obtained. Passage 3 is the same condition with theculture condition of the above-mentioned cardiomyocytes produced by cellsheets.

Both fibroblasts were subjected to primary immunofluorescent stain withGoat polyclonal anti-VCAM-1 antibodies (R&D systems, Minneapolis,Minn.), and were subjected to secondary immunofluorescent stain withAlexa Fluor 488 Donkey anti-goat IgG (Life Technologies, MD, UnitedStates). Subsequently, FACS analysis was conducted at Gallios (BeckmanCoulter, Brea, Calif.), and VCAM-1 positive cell rate was measured(N=3). Calculation of significant difference was carried out byStudent's t-test.

The results of cardiac fibroblasts (NCFs) were shown in FIGS. 7A-C. Itwas found that the percentage of VCAM-1 positive cells in NCFs wasapproximately 60% (FIG. 7A: 66.57%, FIG. 7B: 58.95%, FIG. 7C: 54.73%).Conversely, the percentage of VCAM-1 positive cells in skin fibroblasts(ADFs) was approximately 5%, and it turned out that the percentage ofVCAM-1 positive cells in NCFs is significantly more than that of ADFs(P<0.001).

It was suggested that cardiac fibroblasts containing many VCAM-1positive cells contribute to construction of functional myocardialtissues by proliferating cardiomyocytes derived from mice ES through theexpressing VCAM-1. Further, it was considered that VCAM-1 positivecardiac fibroblasts originate from an outer membrane-derived cell fromthe view point of embryology, and we obtained the suggestion that it iseffective to classify fibroblasts from the view point of embryology, andnot to conduct morphological classification but to conduct functionalclassification as a cell source for constructing a functional tissue.

It is considered that, in NCFs, the majority of the cells that are notexpressing VCAM-1 express CD31 (vascular endothelial cell marker). Thereason for this is as follows: it is known that tissue-resident cardiacfibroblasts are produced from epicardium-derived cells throughepithelial mesenchymal transition (EMT), and also are differentiatedfrom vascular endothelial cells through endothelial mesenchymaltransition (EndMT). Furthermore, as is the case with cardiacfibroblasts, kidney fibroblasts that differentiate from vascularendothelial cells through EndMT are expressing CD31 (J Am Soc Nephrol19:2282-2287, 2008). This may also become one of the bases forsupporting that NCFs are expressing CD31.

From the above, it was clarified that, not skin fibroblasts but cardiacfibroblasts enhance proliferation of mouse embryonic stem cell (mESC)derived cardiomyocytes, and contribute to construction of morefunctional cardiac cell sheets. Moreover, it was indicated that cardiacfibroblasts are more abundantly expressing VCAM-1 compared with skinfibroblasts, and that the VCAM-1 of cardiac fibroblasts play animportant role in proliferation of cardiac cells and construction ofcardiac tissues that are functionally biologically-designed.

Example 2

Example 1 reveals that when a myocardial tissue derived from apluripotent stem cell is constructed, cardiac fibroblasts cause cellproliferation of cardiomyocytes through VCAM-1 which is protein highlyexpressed by the cardiac fibroblasts, prompt beating in the createdwhole myocardial tissue, and significantly improve the functionality.However, it is also revealed that cardiac fibroblasts have aheterozygous character even in a local area named heart, and that allthe cardiac fibroblasts do not necessarily express VCAM-1. Therefore, wecarry out a study to elucidate that, when fibroblasts are not classifiedby the morphological features in a conventional manner but areclassified by protein expressed with molecular biology, to what extentcardiac fibroblasts expressing VCAM-1 (VCFs) should be compounded so asto create a highly functional myocardial tissue.

1. Experimental Method

(1) Animals and Reagents

Wild-type C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan).B6 Cg-Tg (CAG-DsRed*MST) 1Nagy/J mice were purchased from The JacksonLaboratory (Bar Harbor, Me.). All the experimental protocols wereapproved by The Keio University Institutional Animal Care and UseCommittee. The following antibodies were used for immunofluorescentstaining and flow cytometry.

guinea pig monoclonal anti-vimentin (Progen, Heidelberg, Germany),

mouse monoclonal anti-cardiac troponin T (cTnT) (Thermo Scientific,Rockford, Ill.),

rabbit polyclonal anti-Ki67 (Abcam),

Rat monoclonal anti-VCAM-1 (Biotin) (Abcam),

Rat monoclonal anti-CD31 (Abcam),

Rabbit monoclonal anti-VCAM-1 (Abcam).

Secondary antibodies were purchased from ImmunoResearch Laboratories(West Grove, Pa.).

(2) Culture of Mouse ES-Derived Cardiomyocytes

Maintenance, cardiomyocyte differentiation, and purification of mouse EScells (mESC) that express neomycin phosphotransferase genes under thecontrol of α-myosin heavy chain promoter, and that express yellowfluorescent protein (YFP) are conducted in accordance with the method asdescribed previous report (Matsuura K, et al., Biomaterials. 2011;32:7355-7362).

Briefly described, for the purposes of induction to cardiomyocytes andcardiomyocyte purification, 5×10⁴ cells/mL of trypsin-treated ES cells(total 125 mL/flask) were seeded to a spinner flask (IntegraBiosciences, Zizers, Switzerland), and cultured with DMEM supplementedwith 10% FBS for 10 days, and subsequently the differentiated cells weretreated with neomycin for 8 days.

Mouse ES-derived cardiomyocytes (Cor.At) to which puromycin resistancegene and green fluorescent protein (GFP) were introduced under α-myosinheavy chain promoter were purchased from Axiogenesis AG (Cologne,Germany). Mouse ES-derived cardiomyocytes were treated with puromycinfor 2 days, and were cultured in a medium not containing puromycin for 2weeks.

(3) Isolation of VCAM-1 Positive Cardiac Fibroblasts

Cardiac fibroblasts isolated from a one day old wild-type C57BL/6neonatal mouse were cultured, and VCFs were isolated with a magneticcell sorter (Magnetic-activated cell sorting, MACS). The isolated VCFswere re-cultured, and an experiment was carried out after removing deadcells.

(4) Immunofluorescent Staining

Cells were fixed with 4% paraformaldehyde, and immunofluorescentstaining was carried out. The immunofluorescent-stained cells wereanalyzed with a confocal quantification image cytometer CQ1 (YokogawaElectric Corporation, Tokyo, Japan).

(5) Flow Cytometry

Tissues of the heart collected from a wild-type C57BL/6 mouse weredissociated with gentleMACS Octo Dissociator (Miltenyi Biotec, Gladbach,Germany), and homogenized to the cellular level. The obtained cells wereimmunofluorescent-stained, and subsequently analyzed with an S3 cellsorter (BIO-RAD, CA).

(6) Time-Lapse Photography and Analysis

As evaluation of the ability of forming a network, YFP positive EScell-derived cardiomyocytes and cardiac fibroblasts isolated from aDsRed mouse were co-cultured, and the inside of a BZ-9000 fluorescentmicroscope was kept at the concentration of 5% CO2 at 37° C., andtime-lapse observation was carried out for 5 days (Keyence, Osaka,Japan). With respect of the evaluation of the migratory ability of asingle cardiomyocyte, GFP expressing type cardiomyocytes were seeded atthe concentration of 5.45×10⁴ cells/cm²′ and fibroblasts isolated withMACS were seeded at the concentration of 1.36×10⁴ cells/cm², which wereweaker than the concentration at the time when a myocardial tissue wasconstructed, and time-lapse photography was carried out with a BZ-X700fluorescent microscope for 3 days. The photographed time-lapse imageswere analyzed with a Motion Analyzer so as to calculate the totalmigratory distance (mm) of cardiomyocytes for three days of culturing,and the evaluation of the migratory ability was carried out (Keyence).

2. Results

Optimum Compounding Concentration Rates of VFCs and Cardiomyocytes

It was found out that both of VCFs and VCAM-1 negative cardiacfibroblasts (VNCFs) that were isolated and cultured with MACS show afusiform fibroblast-like morphology in phase difference images (FIG.8A). However, by using immunofluorescent staining, while expression ofVCAM-1 protein emitting red fluorescence was observed in almost all thecells in VCFs, expression of VCAM-1 protein was not observed in VNCFs(FIG. 8B).

Subsequently, VCFs and VNCFs collected from cardiac fibroblasts anddermal fibroblasts were co-cultured with cardiomyocytes at eachcompounding ratio (Table 1), the highest mitogenic effect ofcardiomyocytes was observed at Day 5 under the condition where 20% ofVCFs and 80% of cardiomyocytes were seeded (FIGS. 9A and 9B). Inaddition, when the number of fibroblast were calculated by deducting thenumber of triple-positive cells of cTnT/GFP/Hoechst from the totalnumber of Hoechst 33258 positive nuclei in a myocardial tissue at day 5to which VCFs was compounded at 20%, it was revealed that cardiomyocytesexisted at 9.5%, and fibroblasts existed at 90.5% (FIG. 10).

TABLE 1 Compounding ratio of each type of cell VCAM1(+) VCAM1(−) SampleCardiomyocytes NCFs NCFs NCFs ADFs A 80% — — — 20% B 80% 0% 20% — — C80% 4% 16% — — D 80% 8% 12% — — E 80% 12% 8% — — F 80% 16% 4% — — G 80%20% 0% — — H 80% — — 20% —

Evaluation of Migratory Ability of Cardiomyocytes

It was revealed by time-lapse photographing that, when YFP positive EScell-derived cardiomyocytes and cardiac fibroblasts isolated from aDsRed mouse were co-cultured, cardiomyocytes were divided at day 5, anda strong network was constructed (FIG. 11). As mentioned before, cardiacfibroblasts prompt cell division of cardiomyocytes through theexpressing VCAM-1 protein. VCFs and VNCFs isolated with MACS wereco-cultured with GFP expressing type cardiomyocytes, and the totalmigratory distance (mm) of the cardiomyocytes for 3 days of culturingwas calculated by time-lapse photographing, and the evaluation ofmigratory ability was carried out. It was revealed that, when VCFs werecompounded, the number of GFP positive cardiomyocytes grows, and it wassuggested by video analysis that the migratory ability of cardiomyocyteswas significantly high, and took part in forming of a high level network(FIG. 12).

Evaluation of Localization of VCFs in a Biological Heart

It was revealed that, when a myocardial tissue was constructed byco-culturing at the concentration of 20% VCFs and 80% cardiomyocytes,cardiomyocytes grew at the highest level in the myocardial tissue, andobtained high migratory ability (FIGS. 9, 11 and 12). Further, it wasrevealed that 9.5% cardiomyocytes and 90.5% fibroblasts existed in amyocardial tissue created under the above-mentioned condition (FIG. 10).In order to evaluate whether the localization of cardiomyocytes andfibroblasts was different from a biological heart, a heart of a one dayold mouse was collected, and crushed into the cellular level by enzymetreatment, and the evaluation of localization of cardiomyocytes and VCFswere carried out with flow cytometry. It was revealed that VCFs existedat 14.8% in the heart, and that 55.6% of fibroblasts expressed VCAM-1protein (FIGS. 13, 14A and 14B). Moreover, it was revealed thatfibroblasts that express CD31 existed at 3.7% in the heart, and that16.1% of cardiac fibroblasts expressed CD31 protein (FIGS. 13, 14C and14D).

3. Conclusion

The present study revealed that the optimum seeding concentration ofVCFs required to create a high functional myocardial tissue was 20% byevaluating the proliferation level of cardiomyocytes by compounding VCFssorted by a magnetic cell separator (Magnetic-activated cell sorting,MACS) and ES cell-derived cardiomyocytes at each concentration.

Furthermore, it was revealed that the localization of cardiomyocytes andfibroblasts in a myocardial tissue with 20% VCFs at day 5 afterculturing was 9.5% and 90.5%, respectively. When VCFs were seeded at theconcentration of 20% and cardiomyocytes were seeded at the concentrationof 80%, and the evaluation of the migratory ability of cardiomyocyteswas carried out for 3 days with time-lapse photographing, it wasrevealed that VCFs provides cardiomyocytes with high migratory ability,and it was suggested that the provision of high migratory ability takespart in formation of a high level myocardial network in a myocardialtissue.

As a result of flow cytometry, it was revealed that localization ofcardiomyocytes and fibroblasts in a created VCFs-compounded myocardialsheet was greatly different from a biological heart, and it was revealedthat fibroblasts which express Platelet Endothelial Cell AdhesionMolecule-1 (PECAM-1, CD31) other than VCFs are localized in a heart.

INDUSTRIAL APPLICABILITY

By culturing using the cardiac cell culture materials of the presentinvention, functional cardiac tissues are preferably constructed. Thecardiac cells obtained by the culture can be used as regenerativemedicines such as transplantation, or as artificial organ materials suchas cardiac tissue models.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

1. A cardiac cell culture material comprising a fibroblast expressingVCAM-1 protein.
 2. The cardiac culture material according to claim 1used for culturing to construct a cardiac tissue.
 3. The cardiac cellculture material according to claim 1, wherein the fibroblast is acardiac-derived fibroblast.
 4. The cardiac cell culture materialaccording to claim 1, wherein the fibroblast is an epicardial-derivedcell-derived fibroblast.
 5. A cell culture substrate, wherein a wallsurface and/or a bottom surface of the culture substrate having the wallsurface and/or the bottom surface are coated with the cardiac cellculture material according to claim
 1. 6. An artificial organ materialobtained by co-culturing a cardiac cell with the cardiac cell culturematerial according to claim
 1. 7. A method of producing an artificialorgan material comprising a step of co-culturing a cardiac cell with thecardiac cell culture material according to claim
 1. 8. A method forcuring heart diseases by attaching a sheet to a damaged part of theheart wherein, the sheet is constructed from the cardiac cell culturematerial according to claim
 1. 9. A method for constructing a functionalcardiac tissue by attaching a sheet to a damaged part of the heartwherein, the sheet is constructed from the cardiac cell culture materialaccording to claim 1.